Technical Field
The present disclosure relates to compositions and methods for detecting rare nucleic acid molecule mutations in a plurality of nucleic acid molecules.
Description of the Related Art
Mitochondria are multifunctional, essential organelles that play a key role in vital cellular processes such as oxidative phosphorylation (OXPHOS), cellular metabolism, calcium storage and regulation of apoptosis (Scheffler, I. E., Mitochondria, 2nd ed., Wiley-Liss, 2008). Whereas most mitochondrial proteins are encoded in the nucleus, synthesized in the cytosol and subsequently imported into the mitochondria, a subset of genes encoding crucial subunits of the major OXPHOS complexes are encoded within the mitochondria itself on a multi-copy, circular genome.
Maintenance of the mitochondrial genome(s) is crucial for proper organelle function. Accumulated defects, including point mutations, deletions, and rearrangements, can lead to mitochondrial dysfunction and are known to cause a number of mitochondrial disorders (Greaves et al., 2012, J. Pathol. 226:274-286). Large-scale deletions (spanning hundreds to thousands of basepairs) in mtDNA are increasingly associated with a wide variety of pathologies and diseases, including neuromuscular and mitochondrial deletion syndromes (Chinnery, P. F. in Gene Reviews eds. R A. Pagon, T. D. Bird, C. R. Dolan & K. Stephens, 1993), neuropsychiatric disorders (Kato et al., 2011, Neurosci. Res. 69:331-336), Huntington's disease (Horton et al., 1995, Neurology 45:1879-1883), and a growing number of cancers (Lee et al., 2010, Ageing Res. Rev. 9:S47-58). Furthermore, mitochondrial deletions are known to accumulate with age and are thought to be an important driving force in mammalian aging (Trifunovic et al., 2004, Nature 428:417-423; Vermulst et al., 2008, Nat. Genet. 40:392-394; Cortopassi & Arnheim, 1990, Nuc. Acids Res. 18:6927-6933).
While mechanisms for large-scale mtDNA deletions have been proposed (Foury et al, 2004, Cell. Mol. Life. Sci. 61:2799-2811; Krishnan et al., 2008, Nat. Genet. 40:275-279; Song et al., 2011, PLoS Comp. Biol. 7:31002287), they have not been tested in vivo. One of the key difficulties to testing these hypotheses is a lack of sensitive assays that can detect de novo deletions and trace the kinetics of clonal expansion. De novo mtDNA deletions are relatively rare events, occurring with frequencies that are thought to be as low as 1 deletion per million genomes. Furthermore, even though specific deletions are known causes of neuromuscular disorders, this same lack of assay sensitivity has also precluded the use of mtDNA deletions as biomarkers for disease. For example, Kearns-Sayre Syndrome is a mitochondrial deletion disorder caused by accumulation of a large deletion in the mtDNA, typically between nucleotides 8470 and 13446 (the so-called “common deletion”) (DiMauro S. & Hirano, M. in Gene Reviews eds. A. Pagon, T. D. Bird, C. R. Dolan, & K. Stephens, 1993). While the pathogenic deletion is present in all tissues, particularly in skeletal muscle, the measured frequency in blood is low, presumably due to strong selection against dysfunctional genomes in proliferating hematopoietic cells. For this reason, the pathogenic deletion is difficult to detect in patient blood samples using current methods, precluding the use of the deletion as a convenient biomarker for early detection of the disease.